新藤黄酸干预肺腺癌血管生成的研究

［］目的 通过对人脐静脉内皮细胞HUVEC和人肺腺癌A549的培养,检测含新藤黄酸(gambogenic acid,GNA)条件培养基对血管内皮细胞存活率、成管和生长的影响。方法 采用甲基噻唑基四唑(methyl thiazolyltetrazolium,MTT)法和平板克隆实验法研究新藤黄酸对HUVEC存活率和克隆形成率的影响;应用薄层胶原建立血管内皮细胞的二维培养模型,观察新藤黄酸对于血管内皮细胞成管现象的影响;采用细胞划痕愈合和小室迁移实验考察新藤黄酸对 HUVEC的迁移能力影响;Western-blotting检测血管内皮生长因子(VEGF)和缺氧诱导因子(HIF-1α)蛋白的表达。结果 MTT检测结果显示,HUVEC细胞存活率和克隆形成率随GNA剂量增加而降低。新藤黄酸可抑制HUVEC细胞的迁移。还可抑制HUVEC管腔样结构形成。此外,新藤黄酸可下调HUVEC中VEGF和HIF-1α蛋白的表达。结论 新藤黄酸可在体外抑制血管生成,其作用机制可能与抑制肿瘤细胞分泌的缺氧诱导因子(HIF-1α)和血管内皮生长因子(VEGF)有关。     

蜈蚣藻多糖的体外抗血管生成作用

目的探讨蜈蚣藻多糖(GFP)的体外抗血管生成作用。方法应用MTT比色法测定GFP对人脐静脉内皮细胞(HUVEC)存活率的影响;用Millicell小室测定GFP对HUVEC迁移能力的影响;用Matrigel测定GFP对HUVEC管腔形成的影响。结果GFP在5、10、20、50、100mg.L-1浓度下处理HUVEC48h,细胞存活率没有显著性变化;在50、100mg.L-1时能显著性地抑制HUVEC的迁移并呈剂量依赖关系;在100mg.L-1时能显著性地抑制HUVEC的管腔形成。结论GFP能够抑制HUVEC的迁移和管腔的形成。     

雌二醇调控VEGFα表达诱导肺腺癌A549细胞血管形成

目的研究雌二醇(17β-estradiol,E2)在肺腺癌A549细胞微环境中,通过血管内皮生长因子α(vascular endothelial growth factorα,VEGFα)诱导肿瘤血管形成的发生机制。方法将A549细胞分为3组:PBS组、10 nmol·L~(-1) E2组、10 nmol·L~(-1) E2+5μmol·L~(-1)雌激素受体拮抗剂(ICI)组。Western blot和ELISA法检测肺腺癌A549细胞和培养基中VEGFα的表达;MTT法检测人脐静脉内皮细胞(human umbilical vein endothelial cells,HUVECs)的增殖能力;划痕实验检测HUVEC细胞迁移能力;小管生成实验检测HUVEC细胞的体外血管生成能力。结果与对照组(PBS)相比,E2刺激后的A549细胞中VEGFα的表达量增加,而加入ICI后,VEGFα表达量明显下降;同时,E2诱导A549细胞及上清液中VEGFα的表达,促进了HUVEC细胞的增殖、迁移和小管形成能力,ICI则相反。结论 E2通过作用于A549细胞中雌激素受体,刺激肺腺癌微环境中VEGFα的表达,促进肺腺癌血管内皮细胞的增殖、迁移和小管形成能力,诱导肺腺癌血管形成。     

欧芹素乙对人脐静脉内皮细胞的保护作用及对血管内皮生长因子表达的影响

目的:研究欧芹素乙对内皮细胞的保护作用;溶血磷脂酰胆碱(LPC)对人脐静脉内皮细胞株HUVEC细胞中血管内皮生长因子(VEGF)表达的影响以及欧芹素乙的影响.方法:应用四唑盐(MTT)法检测溶血磷脂酰胆碱对HUVEC细胞的毒性作用及欧芹素乙的保护作用;应用基础酶联免疫吸附试验(ELISA)检测各组条件培养基中VEGF蛋白含量;采用RT-PCR法及Reahime PCR方法检测溶血磷脂酰胆碱对VEGF mRNA的表达及欧芹素乙的影响.结果:MTT检测结果显示,溶血磷脂酰胆碱对HUVEC细胞具有较强的生长抑制作用,而欧芹素乙对溶血磷脂酰胆碱所致的细胞增殖抑制具有较好的保护作用.ELISA结果显示,HUVEC细胞暴露于溶血磷脂酰胆碱后,VEGF蛋白含量明显升高;加入欧芹素乙后剂量依赖性地降低VEGF蛋白的表达.RT-PCR结果显示,溶血磷脂酰胆碱可以增加3种VEGF异构体的转录水平,其中VEGF165的表达显著增加,欧芹素乙可剂量依赖性地抑制溶血磷脂酰胆碱引起的VEGF mRNA的高表达.结论:欧芹素乙对溶血磷脂酰胆碱引起的细胞损伤有明显的保护作用;欧芹素乙可抑制溶血磷脂酰胆碱所诱导的HUVEC细胞中VEGF蛋白及VEGF mRNA的高表达,对内皮细胞起到保护作用.     

环磷腺苷葡胺对人脐静脉血管内皮细胞增殖、迁移、管腔形成及凋亡的影响

目的 探索环磷腺苷葡胺（meglumine cyclic adenylate，MCA）对人脐静脉血管内皮细胞（human umbilical vein endothelial cells，HUVEC）增殖、迁移、管腔形成及凋亡的影响。方法 通过显微镜观察、MTT法、划痕修复试验、管腔形成试验、Western blotting观察不同浓度MCA对HUVEC细胞形态、增殖、迁移、管腔形成能力及凋亡相关蛋白表达的影响。结果 显微镜观察发现，随着MCA剂量增加，HUVEC细胞形态由多角形变为卵圆形。MCA可呈浓度依赖地抑制HUVEC增殖、迁移、管腔形成，并且高剂量的MCA还可诱导细胞凋亡，表现为细胞凋亡数目增加，Bax、Cleaved-PARP蛋白表达上调，PARP、Bcl-2蛋白表达下调。结论 MCA可抑制HUVEC细胞增殖、迁移、管腔形成，并诱导细胞凋亡。     

牛蒡子苷对高糖诱导人脐静脉血管内皮细胞损伤的保护作用

目的 探讨牛蒡子苷对高糖诱导的人脐静脉血管内皮细胞(HUVECs)损伤的保护作用及其机制.方法 用高糖诱导HUVECs,建立内皮细胞损伤模型,经牛蒡子苷处理后,用MTT法、划痕试验及Matrigel法分别检测牛蒡子苷对内皮细胞增殖、迁移及血管腔形成能力的影响;用Western blotting法检测牛蒡子苷对低氧诱导因子-1α(HIF-1α)、血管内皮生长因子A(VEGF-A)及Akt蛋白表达的影响.结果 与模型组比较,牛蒡子苷组HUVECs的增殖、迁移及血管腔形成能力明显降低;HIF-1α、VEGF-A和Akt蛋白的表达亦显著下调.结论 牛蒡子苷可能通过阻断Akt-HIF-1α-VEGF-A信号通路来抑制高糖诱导的HUVECs的增殖、迁移及血管新生.     

PI3K/Akt抑制剂CCT128930抑制人脐静脉血管内皮细胞增殖与血管生成的实验研究

目的探讨一类新的PI3K/Akt抑制剂CCT128930对人脐静脉血管内皮细胞(HUVEC)增殖与血管生成的影响。方法采用MTT法检测CCT128930对HUVEC存活的影响;流式细胞术分析细胞周期变化;AnnexinⅤ-FITC试剂盒检测细胞凋亡;体外小管形成实验观察CCT128930对HUVEC体外小管形成的影响;免疫印迹法检测蛋白表达水平。结果CCT128930可通过阻滞细胞于G1期而抑制HUVEC的增殖,且抑制作用呈剂量依赖性,但对HUVEC的凋亡无影响;HUVEC经CCT128930处理后,体外小管形成能力受到明显抑制;低浓度的CCT128930抑制内皮细胞中VEGF的表达,但对Akt的磷酸化水平无影响。结论 CCT128930能够抑制HUVEC的增殖与血管生成,其抑制血管生成活性可能与其调控VEGF的表达水平相关。     

革皮氏海参皂苷抑制血管新生作用

目的以革皮氏海参中分离得到的三萜皂苷ho-lothurin A1(HA)和24-dehydroechinoside A(DA)为研究对象,比较研究其抗血管新生的作用。方法采用MTT法和AO/EB染色法检测不同剂量HA和DA对人脐静脉内皮细胞(HUVEC)增殖和凋亡的影响;采用小管形成实验观察HA和DA对HUVEC分化形成小管能力的影响;细胞黏附实验比较研究HA和DA对HUVEC与肝癌细胞HepG2的黏附能力的影响;鸡胚绒毛尿囊膜(CAM)血管新生模型,观察HA和DA抑制CAM血管新生的情况。结果 HA和DA均具有抑制HUVEC增殖的活性(48 h的IC50值分别为4.80、3.82μmol.L-1),并能促进内皮细胞的凋亡;在体外能抑制内皮细胞小管形成,可抑制HUVEC与HepG2细胞间(P<0.01)的黏附,能使CAM新生血管明显减少,其中DA抑制血管新生活性优于HA。结论海参皂苷HA和DA具有抑制血管新生的作用,其活性与其结构相关。     

PI3Kα抑制剂BYL719抑制脐静脉血管内皮细胞增殖及血管生成的研究

目的 探讨PI3Kα抑制剂BYL719对人脐静脉血管内皮细胞(human umbilical vein endothelial cells,HUVEC)增殖与血管生成的影响。方法 采用MTT法测定BYL719对HUVEC活力的抑制作用;用流式细胞术检测BYL719对其细胞周期的影响;体外小管形成实验观察BYL719对血管生成的影响;western blot检测Akt、p-Akt、cyclinD1和CDK4蛋白表达水平的变化。结果 BYL719抑制了HUVEC中Akt的磷酸化水平;BYL719作用细胞后,HUVEC的细胞活力显著降低;BYL719能够通过下调cyclinD1和CDK4的表达而阻滞细胞于G1期,进而抑制HUVEC的增殖;HUVEC经BYL719处理后,体外小管形成能力受到显著抑制。结论 BYL719降低了HUVEC中Akt的磷酸化水平,能够抑制HUVEC的细胞增殖和血管生成。     

海洋溴酚BTDE抑制内皮细胞血管生成的研究

目的 探究海洋溴酚化合物双（2,3,6-三溴-4,5-二羟基苄基）醚（BTDE）处理肿瘤细胞和肿瘤相关巨噬细胞所获得的条件培养基对内皮细胞血管生成的影响。方法 MTT法检测BTDE对肿瘤相关巨噬细胞RAW264.7增殖的影响；Transwell实验检测BTDE对细胞迁移和侵袭的影响；明胶酶谱法检测BTDE对细胞分泌的基质金属蛋白酶9（MMP9）活性的影响；Western Blot检测BTDE对细胞中β-catenin、VEGF表达的影响；体外获取BTDE处理肺癌A549后的条件培养基（BTDE/A549-CM）和处理肿瘤相关巨噬细胞RAW264.7后的条件培养基（BTDE/RAW264.7-CM），采用Transwell和Tube formation实验检测条件培养基对人脐静脉内皮细胞HUVEC迁移和成管的影响。结果 BTDE抑制RAW264.7细胞的迁移、侵袭和分泌的MMP9活性；BTDE/A549-CM和BTDE/RAW264.7-CM抑制HUVEC细胞的迁移和血管生成，血管内皮细胞的血管生成率在5 μM和10 μM BTDE/A549-CM处理下为75.0％和23.8％，在2.5 μM，5 μM和?10 μM BTDE/RAW264.7-CM处理下为54.1％，35.69％和18.8％。 结论 海洋溴酚BTDE处理肺癌细胞A549和肿瘤相关巨噬细胞RAW264.7后的条件培养基能够抑制血管内皮细胞HUVEC的迁移和血管生成，提示BTDE有潜力发展为临床抗肿瘤血管生成治疗药物。     

岩藻糖基化海参硫酸软骨素抑制肿瘤血管新生作用的研究

目的从美国肉参(Isostichopus badionotus)中分离纯化岩藻糖基化海参硫酸软骨素(sea cucumber chondroitin sulfate,SC-CHS),体外研究其对肿瘤诱导血管生成的抑制作用。方法采用MTT法检测SC-CHS对95D细胞增殖活性的影响;小管形成实验研究SC-CHS对脐静脉内皮细胞(HUVEC)小管形成能力的抑制作用;采用鸡胚尿囊膜(CAM)新生血管模型,研究其对血管生成的影响;通过RT-PCR和WesternBlot法检测其对肿瘤诱导血管生成相关因子缺氧诱导因子(hypoxia inducible factor-1α,Hif-1α)和血管内皮生长因子(vascular endothelial growth factor,VEGF)mRNA和蛋白的表达。结果结果显示SC-CHS能显著抑制95D细胞的增殖,能抑制HUVEC的小管形成作用和CAM血管新生。RT-PCR和Western Blot检测结果表明,高剂量SC-CHS能显著降低95D细胞中Hif-1α和VEGF mRNA的表达,减少VEGF蛋白的合成。结论SC-CHS体外能显著抑制肿瘤血管生成,这可能是通过影响肿瘤细胞中VEGF的合成发挥作用。     

Saikosaponins-b suppresses tumor growth and angiogenesis of hepatocellular carcinoma by regulating VEGF/ERK/HIF-1α signal pathway

OBJECTIVE Angiogenesis therapy has attracted interest as a potential treatment for hepatocellular carcinoma(HCC).In this study,we investigated the anti-proliferative activities and antiangiogenesis effects of saikosaponins(SS)-b on hepatocellular carcinoma(HCC)and its regulation on VEGF/ERK/HIF-1 αsignal pathway.METHODS H22 hepatoma-bearing mice model and HepG-2 cells were used to study the anti-tumor and anti-angiogenesis effects of SS-b in vivo and in vitro.Pathological change of tumor tissue was observed by HE staining,the microvascular changes were detected by immunohistochemical method.The effects of SS-b on angiogenesis were examined by using the chick embryo chorioallantoic membrane(CAM)model.The effects of SS-b on proliferation,migration and invasion were investigated by MTT assay,scratch wound healing assay and transwell assay inhuman umbilical vein endothelial cell(HUVEC)and HepG2 cells in vitro.Vascular endothelial growth factor(VEGF),matrix metalloproteinase-2/9(MMP-2/9),hypoxia-inducible factor-1α(HIF-1α)expression and the phosphorylation of extracellular regulated kinase(ERK)were analyzed using RT-PCR and Westernblot.RESULTS SS-b effectively inhibited the tumor growth of H22 mice in vivo.The inhibitory rate of tumor was 49.1%,50.7%,66.1%in SS-b 5,10 and 20 mg·kg-1group respectively.HE staining results showed that SS-b induced tumor necrosis and nuclear dissolution in H22 mice.Moreover,SS-b also reduced the number of microvessels of tumor tissue in H22 mice significantly and suppressed the angiogenesis of CAM induced by b-FGF.SS-b had an obvious inhibitory effect on cell proliferation,migration and invasion of HUVEC cells and HepG-2 cells.These effects were associated with downregulation of the expression of MMP2/9 and suppression of VEGF/ERK/HIF-1αsignaling in H22 mice and Hep-G2 cells.CONCLUSION Our findings showed that SS-b exerts anti-tumor effects by inhibiting tumor angiogenesis via regulating VEGF/ERK/HIF-1α signal pathway in vivo and in vitro.     

卡立泊来德对K562细胞诱导的血管生成的影响

目的研究卡立泊来德(cariporide)处理对K562细胞诱导的血管生成能力的影响。方法应用MTT检测K562细胞上清液对脐静脉内皮细胞增殖能力的影响;transwell检测K562细胞上清液对脐静脉内皮细胞迁移能力的影响;基质胶血管形成法检测K562细胞上清液对脐静脉内皮细胞体外血管形成能力的影响;激光扫描共聚焦显微镜测定K562细胞的细胞内的pH;酶联免疫吸附实验(ELISA)检测K562细胞上清中血管内皮生长因子的表达水平。结果cariporide处理可以明显降低K562细胞上清液对脐静脉内皮细胞增殖,迁移和体外成管能力的诱导;cariporide处理后K562细胞的细胞内pH明显下降,分泌VEGF能力也受到抑制。结论 cariporide能抑制K562细胞的血管生成诱导能力,这种抑制是通过细胞内pH下降以及VEGF分泌减少引起的。     

Dauricine inhibits insulin-like growth factor-I-induced hypoxia inducible factor 1α protein accumulation and vascular endothelial growth factor expression in human breast cancer cells

Aim:

To investigate the effects of dauricine (Dau) on insulin-like growth factor-I (IGF-I)-induced hypoxia inducible factor 1α (HIF-1α) and vascular endothelial growth factor (VEGF) expression in human breast cancer cells (MCF-7).

Methods:

Serum-starved MCF-7 cells were pretreated for 1 h with different concentrations of Dau, followed by incubation with IGF-I for 6 h. HIF-1α and VEGF protein expression levels were analyzed by Western blotting and ELISA, respectively. HIF-1α and VEGF mRNA levels were determined by real-time PCR. In vitro angiogenesis was observed via the human umbilical vein endothelial cell (HUVEC) tube formation assay. An in vitro invasion assay on HUVECs was performed.

Results:

Dau significantly inhibited IGF-I-induced HIF-1α protein expression but had no effect on HIF-1α mRNA expression. However, Dau remarkably suppressed VEGF expression at both protein and mRNA levels in response to IGF-I. Mechanistically, Dau suppressed IGF-I-induced HIF-1α and VEGF protein expression mainly by blocking the activation of PI-3K/AKT/mTOR signaling pathway. In addition, Dau reduced IGF-I-induced HIF-1α protein accumulation by inhibiting its synthesis as well as by promoting its degradation. Functionally, Dau inhibited angiogenesis in vitro. Moreover, Dau had a direct effect on IGF-I-induced invasion of HUVECs.

Conclusion:

Dau inhibits human breast cancer angiogenesis by suppressing HIF-1α protein accumulation and VEGF expression, which may provide a novel potential mechanism for the anticancer activities of Dau in human breast cancer.     

Chrysin inhibits lipopolysaccharide-induced angiogenesis via down-regulation of VEGF/VEGFR-2(KDR) and IL-6/IL-6R pathways

The relationship between chrysin and inflammation-induced angiogenesis remains unclear. The aim of this study was to evaluate the suppressive effects of chrysin on lipopolysaccharide (LPS)-induced angiogenesis in chicken chorioallantoic membrane (CAM) as well as in human umbilical endothelial cells (HUVEC). The IN VIVO CAM model was applied to evaluate the percentage of new vessels formation, followed by measuring endothelial migration and tube formation in HUVEC cultures. The mechanisms of the suppressive effect of chrysin on LPS-induced angiogenesis, in terms of VEGF, VEGF receptors (VEGFR), interleukin 6 (IL-6) and IL-6 receptor gene expressions, were analyzed by Western blot, ELISA cytokine assay, and quantitative real time PCR. The results showed that chrysin (10(-8) - 10(-5) M) inhibited LPS-induced CAM neovascular density. There was a significant down-regulation of VEGF and VEGFR-2 (KDR) but not VEGFR-1 (Flt-1) gene expression by chrysin in LPS-treated HUVEC cultures. Besides, chrysin concentration-dependently inhibited the auto-regulation loop of IL-6/IL-6R in LPS-treated HUVEC cells. We conclude that chrysin suppresses both IN VITRO and IN VIVO LPS-induced angiogenesis.     

N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide promotes vascular endothelial cell angiogenesis and migration in the absence of serum and FGF-2

Aim:

To investigate the effect of N-benzyl-5-phenyl-1H-pyrazole-3-carboxamide (BPC) on angiogenesis in human umbilical vein endothelial cells (HUVECs).

Methods:

Capillary-like tube formation on matrigel and cell migration analyses were performed in the absence of serum and fibroblast growth factor (FGF-2). Reactive oxygen species (ROS) were measured using a fluorescent probe, 2′, 7′- dichlorodihydrofluorescein (DCHF). The nitric oxide (NO) production of HUVECs was examined using a NO detection kit. Morphological observation under a phase contrast microscope, a viability assay using 3-[4, 5-dimethylthiazol-2-yl]-2, 5-diphenyl-tetrazolium (MTT) and a lactate dehydrogenase (LDH) activity analysis by a detection kit were performed to evaluate the toxicity of BPC on HUVECs in the presence of serum and FGF-2. The level of hypoxia-inducible factor 1α (HIF-1α) and the release of vascular endothelial growth factor (VEGF) were measured by Western blot and ELISA, respectively.

Results:

In the absence of serum and FGF-2, cells treated with BPC (5-20 μmol/L) rapidly aligned with one another and formed tube-like structures within 12 h. In the presence of serum and FGF-2, cells treated with BPC for 24, 48 and 72 h had no changes in morphology, viability or LDH release compared with the control group. Cell migration in the BPC-treated group was significantly increased compared with the control group. During this process, NO production and ROS level were elevated dramatically, and the levels of HIF-1α and VEGF were increased dependent on the generation of ROS.

Conclusion:

BPC most effectively promoted angiogenesis and migration in HUVECs in the absence of FGF-2 and serum.     

芸香宁碱抑制血管生成作用的研究

目的：研究芸香宁碱对血管生成的抑制作用,并研究其初步的作用机制。方法用人脐静脉内皮细胞（HUVEC）的生长、迁移实验及体内动物模型－鸡胚绒毛尿囊膜法（CAM 法）研究化合物的体内外抗血管生成作用;用酶联免疫吸附法检测芸香宁碱在非凋亡剂量时对肿瘤细胞培养上清液中 VEGF 蛋白分泌量的影响。结果芸香宁碱对血管内皮细胞具有优先抑制作用,对 HUVEC 的半数抑制剂量为（33．2±0．4）μmol·L －1,而对其他肿瘤细胞的 IC50值均大于这一数值;芸香宁碱在体外能够明显抑制内皮细胞的迁移和对细胞外基质黏附作用,并呈剂量依赖性,体内实验显示30μmol·L －1剂量浓度时显著抑制 CAM新生血管形成;芸香宁碱在15μmol·L －1和30μmol·L －1的浓度剂量时显著抑制人肝癌 HepG2细胞培养上清液中VEGF 蛋白的分泌,具有显著性差异。结论芸香宁碱在非凋亡浓度剂量具有抑制新生血管形成作用,其机制与抑制血管内皮细胞增殖以及抑制肿瘤细胞表达 VEGF 有关。     

Inhibition of angiogenesis involves in anticancer activity of riccardin D, a macrocyclic bisbibenzyl, in human lung carcinoma

Riccardin D is a novel macrocyclic bisbibenzyl compound extracted from Chinese liverwort plant Dumortiera hirsuta. Our previous studies showed that riccardin D is a DNA topo II inhibitor and has therapeutic potential for treatment of cancers. In this combined in vitro and in vivo study, we examined the inhibitory effects of riccardin D on tumor angiogenesis and the subsequent effect of anticancer activity was evaluated. Incubation with riccardin D weakly inhibited the proliferation of human umbilical vascular endothelial cells (HUVEC) as estimated by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. The scratch wound experiment showed that riccardin D effectively decreased the motility and migration of HUVEC cells. Riccardin D inhibited the formation of capillary tube as demonstrated by decrease of branch points formed by HUVEC cells on 3-D Matrigel. We examined the levels of angiogenic factors including vascular endothelial growth factor (VEGF), VEGF receptor 2, epidermal growth factor receptor (EGF receptor), and matrix metalloproteinase (MMPs) in HUVEC cells. The expressions of VEGF, phospho-VEGF receptor 2, EGF receptor and MMP-2 were significantly reduced by riccardin D as estimated by Western blot assay and real-time quantitative PCR analysis. The decrease of VEGF was also detected in riccardin D-treated human lung cancer H460 cells. The anticancer activity of riccardin D was then evaluated in a mouse model in which riccardin D delayed the growth of H460 xenografts without obvious toxicity to animals after three weeks injection. To evaluate the role of antiangiogenesis of riccardin D in mice, CD34 immunohistochemical staining was employed to analyze the mean vascular density in H460 xenograft tissues. The number of blood vessels was significantly decreased after riccardin D treatment. These results suggest that riccardin D display the inhibitory effect on growth of human lung carcinoma cells and that the inhibition of angiogenesis may involve in anticancer activity of riccardin D.     

Berberine inhibits HIF-1alpha expression via enhanced proteolysis

We have studied the antiangiogenic property of berberine. We showed that berberine could directly inhibit in vitro human umbilical vein endothelial cell (HUVEC) tube formation and migration. In addition, to determine whether berberine could influence the cross-talk between the gastric adenocarcinoma cell line SC-M1 and vascular endothelial cells, we performed modified confrontation culture experiments and showed that berberine (7.5 microM, 16 h) could inhibit the capacity of hypoxic SC-M1 cells to stimulate HUVEC migration. These results demonstrated berberine's antiangiogenic property and its clinical potential as an inhibitor of tumor angiogenesis. Parallel Western blot analyses revealed that berberine prevented hypoxic SC-M1 cultures from expressing vascular endothelial growth factor (VEGF) and hypoxia-inducible factor (HIF)-1alpha, two key factors in mediating tumor angiogenesis. However, overexpression of HIF-1alpha in SC-M1 cells dramatically reversed the inhibitory effect of berberine on SC-M1-induced in vitro HUVEC migration. These data indicated that HIF-1alpha repression is a critical step in the inhibitory effect of berberine on tumor-induced angiogenesis. Northern blot analyses plus pulse-chase assays revealed that berberine did not down-regulate HIF-1alpha mRNA but destabilized HIF-1alpha protein. We found that berberine-induced HIF-1alpha degradation was blocked by a 26S proteasome inhibitor. Moreover, immunoprecipitation and Western blot analyses showed that berberine increased the lysine-acetylated HIF-1alpha in hypoxic SC-M1 cultures. These data indicated that a proteasomal proteolytic pathway and lysine acetylation were involved in berberine-triggered HIF-1alpha degradation. In conclusion, our data provided molecular evidence to support berberine as a potent antiangiogenic agent in cancer therapy.